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Figure 1. Overview of the oocyte injection experiments. MI, AI, and TI indicate metaphase, anaphase, and telophase of meiosis I, respectively. IK, interkinasis; MII, metaphase of meosis II. Time after GVBD is indicated in hours.
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Figure 2. Localization of Bub1dn-YFP on kinetochores. Bub1dn-YFP and histone H2B-RFP mRNAs were coinjected into GV-stage oocytes, kept in dbcAMP for 1 h, released, and fixed 5â6 h after GVBD (1st meiosis) or overnight culture (2nd meiosis). (Left) The whole oocyte is shown in an overview, with chromosomes in red (histone H2B-RFP) and Bub1dn-YFP in green. The position of the PB is marked. (Right) Chromosomes are enlarged, and the signals from histone H2B-RFP and Bub1dn-YFP individually, as well as overlaid (merge). Note the dotted appearance of the Bub1dn-YFP signal around the chromosomes suggesting a kinetochore localization. Bar, 10 μm.
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Figure 3. The effect of Bub1dn injection on meiosis I and II in mouse oocytes. (A) Time-lapse video microscopy. Uninjected, and Bub1dn-injected oocytes were kept 1 h in dbcAMP before they were released from the prophase block. 3 h after GVBD, the oocytes were placed under the microscope in a 37°C heating box. Pictures were taken every 45 min for 12 h. Red arrows indicate the extruded PB. Time points (h) after GVBD are indicated. Bar, 50 μm. (B) Bub1dn disrupts the SAC in meiosis II. Uninjected, and Bub1dn-injected oocytes were cultured for 14â15 h after GVBD and transferred into individual drops of medium containing nocodazole, or strontium, or both, and then cultured for an additional 3â4 h. Oocytes were fixed and stained with PI (red) and an anti-tubulin antibody, followed by an FITC-labeled secondary antibody (green; a and d), or with DAPI alone (b and e). The position of the PB is marked, and the oocyte chromosomes are boxed. (c and f) The DAPI stained chromosomes from b and e were enlarged by 2.6 times. Bar, 10 μm. (C) Single cell H1/MBP kinase assay. Total H1 and MBP kinase activities were assayed in whole cell lysates prepared from uninjected, GV-stage, and Bub1dn-injected oocytes. The autoradiograph is shown.
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Figure 4. The effect of Mad2ÎC and BubR1d injection on meiosis I and II in mouse oocytes. (A) Time-lapse video microscopy. Mad2ÎC or BubR1d injected oocytes were treated as in Fig. 3 A. Pictures were taken every 30 min for 12 h. Red arrows indicate the extruded PB. Time points (h) after GVBD are indicated. Bar, 50 μm. (B) Immunofluorescence staining of Mad2ÎC (left) or BubR1d-injected (right) oocytes. Mad2ÎC or BubR1d-injected oocytes were kept 1 h in dbcAMP before they were released from the prophase block. 20 h after GVBD, the oocytes were fixed, chromosomes stained with PI (red), and the meiotic spindles visualized using an anti-tubulin antibody, followed by an FITC-labeled secondary antibody (green). Note the extruded PB, and the chromosomes aligned on a metaphase plate with an intact spindle apparatus, indicating a CSF-arrest. Bar, 10 μm. (C) Activation of Mad2ÎC and BubR1d-injected oocytes. Uninjected, and Mad2ÎC- or BubR1d-injected oocytes were cultured for 14â15 h after GVBD and transferred into individual drops of medium containing nocodazole, or strontium, or both, and then cultured for an additional 3â4 h. Chromosomes were stained with DAPI. The position of the PB is marked. The oocyte nuclei are boxed, and shown enlarged in the insets. Bar, 10 μm.
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Figure 5. Localization of Cdc20-YFP. Cdc20-YFP and histone H2B-RFP were coinjected into GV-stage oocytes, kept in dbcAMP for 1 h, released and fixed 6 h after GVBD (1st meiosis), 9â10 h after GVBD (1st anaphase and 1st telophase), or after overnight culture (2nd meiosis). (Left) The whole oocyte is shown in an overview, with chromosomes in red (histone H2B-RFP) and Cdc20-YFP in green. The position of the PB is marked in the metaphase II-arrested oocytes. (Right) The chromosomes are enlarged, and the signals from histone H2B-RFP and Cdc20-YFP are shown individually, as well as overlaid (merge). Bars, 10 μm.
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Figure 6. The effect of Cdc20-GR and -4AV injection into mouse oocytes. (A) Time-lapse video microscopy. Uninjected, or oocytes injected with Cdc20-GR or -4AV were treated as in Fig. 3 A. Red arrows indicate the extruded PB. Time points (h) after GVBD are indicated. Bar, 50 μm. (B) Immunofluorescence staining of control (left), or Cdc20-GR injected (middle), or Cdc20-4AV injected (right) oocytes. Oocytes were treated as in Fig. 4 B. Note the extruded PB, and the chromosomes aligned on a metaphase plate with an intact spindle apparatus. (C) The localization of Cdc20 is affected by checkpoint protein mutants. Cdc20-YFP and histone H2B-RFP mRNA and Bub1dn- or Mad2ÎC- or BubR1d-injected oocytes were treated as above, and fixed 5â6 h (1st meiosis) after GVBD, or after overnight culture (2nd meiosis). The histone H2B-RFP and Cdc20-YFP signals are shown individually, as well as overlaid (merge). Note the dotted appearance of the Cdc20-YFP signal around the chromosomes, suggesting a kinetochore localization, in the Mad2ÎC coinjected oocytes, which is weaker in the BubR1d coinjected, and seems absent from the Bub1dn coinjected oocytes. (D) The localization of the Cdc20-GR or -4AV YFP-fusion proteins. Cdc20-GR-YFPâ or Cdc20-4AV-YFPâ and H2B-RFPâinjected oocytes were treated as above, and fixed 6 h (1st meiosis) after GVBD, or after overnight culture (2nd meiosis). Note the dotted appearance of the Cdc20-4AV-YFP signal around the chromosomes, suggesting a kinetochore localization, which is not detectable in Cdc20-GR-YFPâinjected oocytes. Bars: (BâD) 10 μm.
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Figure 7. The effect of Cdc20-4AVGR injection into mouse oocytes. (A) Time-lapse video microscopy. Uninjected, or oocytes injected with the Cdc20-4AVGR mutant were treated as in Fig. 3 A. Red arrows indicate the extruded PB. The oocyte marked by the green box failed, despite several attempts, to extrude its PB. The pink arrow indicates the spot where the PB should have been extruded. The oocyte marked by the blue box, however, divided abnormally and extruded two PBs, indicated by the light blue arrow. Time points (h) after GVBD are indicated. Bar, 50 μm. (B) Immunofluorescence staining of uninjected, and Cdc20-4AVGRâinjected oocytes. Oocytes were treated as in Fig. 4 B. (Left) The whole oocyte is shown in an overview. (Right) The area of the spindle is enlarged, and the DNA and tubulin staining are shown individually, as well as overlaid (merge). The uninjected oocyte extruded the PB, and the chromosomes are aligned on a metaphase plate with an intact spindle apparatus. Examples of the various phenotypes of Cdc20-4AVGRâinjected oocytes are shown below. In bâd, the oocytes did not extrude their first PB. The abnormal spindles in c and d are marked with red arrows. In e, an oocyte with an interphase nucleus in the absence of any extruded PB is shown. In f, a typical example of an oocyte with one PB extruded is shown. In g, an oocyte is shown with two extruded PBs. Bars, 10 μm. (C) Activation of Cdc20-4AVGRâinjected oocytes. The oocytes were treated as in Fig. 4 C. The position of the PB is marked. The oocyte nuclei are boxed, and shown enlarged in the insets. (D) Single cell H1/MBP kinase assay. Total H1 and MBP kinase activities were assayed in whole cell lysates prepared from uninjected, GV-stage, and Cdc20-, Cdc20-4AVâ, and Cdc20-4AVGRâinjected oocytes. The autoradiograph is shown.
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Figure 8. Cdc20-4AVGR mutant induces sister chromatid separation. (A) Chromosome spreads were prepared from noninjected, or Cdc20-4AVGRâinjected oocytes after overnight culture. The chromosomes were stained using a 2% Giemsa solution. In the insets, chromosomes are shown that were found outside the main field. a, uninjected in 1st meiosis (6 h after GVBD); b, uninjected in 2nd meiosis; c and d, Cdc20-4AVGRâinjected oocytes with the PB extruded; e and f, Cdc20-4AVGRâinjected oocytes with no PB extruded. Bar, 10 μm. (B) Quantification of A. On the x axis are the numbers of chromosomes counted per oocyte, on the y axis the number of oocytes. a, control; b, Cdc20-4AVGRâinjected oocytes with PB extruded; c, Cdc20-4AVGRâinjected oocytes with no PB extruded. (C) Time-lapse video microscopy. The Cdc20-4AVGR construct was coinjected into oocytes either with (a) cyclin B1N170-RFP (a 170âamino acid NH2-terminal fragment of cyclin B1 fused to RFP) or with (b) Pds1-RFP. The oocytes were kept in dbcAMP for 1 h, released 4 h after GVBD, and observed by differential interference contrast, and fluorescence time-lapse video microscopy. Pictures were taken every hour (BN170-RFP) or every 45 min (Pds1-RFP). The complete series is shown. Time points (h) after GVBD are indicated. Bars, 50 μm.
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